ES2350431A1 - Vineyard osmotolerante cect 13015 year and its application in the production and improvement of sweet wines and the obtaining of bioethanol. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Cement 13015 osmotolerant wine yeast and its application in the production and improvement of sweet wines and the production of bioethanol, where a strain of saccharomyces cerevisiae cect 13015 is applied for the production of wines, especially sweet wines, as well as for the production of bioethanol from sugary substrates. This strain has been isolated from a fermented grape must by pedro ximºnez from the d.o. Montilla-moriles, and has been selected for its rapid fermentation kinetics, for its excellent organoleptic qualities, as well as for its high ethanol production. (Machine-translation by Google Translate, not legally binding)

Description

Osmotolerant wine yeast CECT 13015 and its application in the production and improvement of sweet wines and obtaining of bioethanol.

The present invention aims at the application of a strain of Saccharomyces cerevisiae CECT 13015 for the production of wines, especially sweet wines, as well as for the production of bioethanol from sugary substrates. This strain has been isolated from a Pedro Ximénez fermented grape must from the DO Montilla-Moriles, and has been selected for its rapid fermentation kinetics, for its excellent organoleptic qualities, as well as for its high ethanol production.

Prior art

In some warm regions, with specials climatic characteristics, special sweet wines are obtained improving the quality of the grapes harvested at maturity, through a natural drying process by direct exposure of the Grapes in the sun for a few days. This happens in wine regions with a heliothermal index of 2597 ° C or higher, located in several Mediterranean countries such as Spain, Greece, Cyprus, Italy and Turkey, where special dessert wines are produced by this method to from different grape varieties, although they are mainly used Muscat varieties. In Andalusia (Spain) and particularly in the areas of Montilla-Moriles, Jerez and Málaga, are In addition to the Muscat grape, the Pedro Ximénez grape.

The must obtained from the Pedro Ximénez grapes at medium maturity has a sugar content of around 25ºBx and after the process of passification it presents a content that reaches up to 45ºBx, equivalent to 500 g / L, (Franco M, Peinado RA, Medina M, Moreno J. 2004. Off-vine grape drying effect on volatile compounds and aromatic series in must from Pedro Ximénez grape variety. J Agrie Food Chem 52 : 3905-3910).

Saccharomyces cerevisiae is a saccharophilic fungus and in its habitat is found with high concentrations of sugars. However, when used to ferment musts with a high concentration of sugars, a series of problems occur, such as slow fermentation stops and fermentation, which can favor the development of undesirable microorganisms, which increase volatile acidity and produce low quality wines ( Caridi A., Crucitti P., Ramondino D. 1999. Winemaking of must at high osmotic strength by thermotolerant yeast. Biotechnol Lett , 21 : 617-620). In Salmon JM, Mauricio JC. 1994. Relationship between sugar uptake kinetics and total sugar consumption in different industrial Saccharomyces cerevisiae strains during alcoholic fermentation. Biotechnol Lett 16 : 89-94 describes a lower activity in the transport of glucose in yeasts that ferment media with a high concentration of sugars. In order to avoid these problems, some special sweet wines have been traditionally made in certain areas by mixing the must of grape with wine alcohol. This fact causes problems in the cataloging of the product in the market and for the final consumer, since it has not been elaborated through a fermentation process. A possible solution could be the realization of partial fermentations directed with isolated yeasts and selected from raisins.

The energy crisis caused by the dependence on fossil fuels, as well as the contaminating effect they have on the environment, together with their contribution to the greenhouse effect, makes it necessary to search for alternative or complementary renewable energy sources, whose net balance in the emission of CO2 is lower (Cot M, Loret MO, François J, Benbadis L. 2007. Physiological behavior of Saccharomyces cerevisiae in aerated fed-batch fermentation for high level production of bioethanol. FEMS Yeast Res 7 : 22-32). One of these energy sources is bioethanol obtained from plant carbohydrates (Liang L, Zhang Y, Zhang L, Zhu M. Liang S, Huang Y. 2008. Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses J Ind Microbiol Biotechnol 35 : 1605-1613), which is particularly interesting as fuel for motor vehicles either alone or mixed with other fuels. Ethanol is obtained through a three-phase process:

i)

obtaining a solution of sugars fermentable,

ii)

alcoholic fermentation of said solution, Y

iii)

distillation and dehydration of ethanol. The total process efficiency is strongly related to the inverted energy, particularly in the phase of distillation.

Different sources of fermentable carbohydrates have been used, depending on their abundance or availability in the immediate environment or by-products or residues of local agriculture and industry. In an unconscious way, substrates such as corn and wheat have been used, without assessing the negative impact that the deviation of these grains towards bioethanol production has had on human food. These substrates, rich in starch or carbohydrates, not directly assimilable by fermentative microorganisms, require expensive acid or enzymatic pre-treatments or heating to convert non-assimilable carbohydrates into fermentable sugars (Leipper KA, Schlee C, Tebble I, Stewart GG. 2006 The fermentation of beet sugar syrup to produces bioethanol. J Inst Brew 112 : 122-133.) Which increase the production costs and the energy required to obtain ethanol. The use of substrates that have high content in fermentable sugars (molasses or juices from the process of extracting cane sugar or beet) does not require special treatments, so the cost (economic and energy) of the process will be lower.

On the other hand, it is known that the higher the ethanol content of a mixture, the lower the energy consumption necessary for its separation (Bertolini MC, Ernandes JR, Laluce C. 1991. New yeast strains for alcoholic fermentation at higher sugar concentration. Biotechnol Lett 13 : 197-202). Thus, the higher the concentration in ethanol obtained in the fermentation phase, the lower the energy invested in distillation. Consequently, the optimization of the fermentation phase with respect to the use of osmotolerant microorganisms that produce high concentrations of ethanol from substrates with high sugar content is revealed as an interesting alternative to increase the efficiency of the process of obtaining bioethanol.

Saccharomyces cerevisiae yeast is the most commonly used microorganism in alcoholic fermentation, although currently the Zymomonas mobilis bacteria is also being used because it has a number of advantages over S. cerevisiae (Zaldivar J, Nielsen J, Olsson L. 2001. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 56 : 17-34).

As previously mentioned S. cerevisiae is a saccharophilic fungus and in its habitat is found with high concentrations of sugars. However, when used to ferment musts with a high concentration in sugars, a number of problems occur, such as slow fermentation stops and fermentation (Caridi et al ., 1999). Salmon and Mauricio (1994) have described a lower activity in the transport of glucose in yeasts that ferment media with a high concentration of sugars.

To avoid these problems, one possible solution could be the realization of fermentations directed with isolated and selected yeasts from very sugary media such as raisins, which reach more than 400 g / L of fermentable sugars. Normally, yeast strains involved in industrial processes have an osmotolerance limited, so fermentations are made from sugar concentrations below 20% (w / v). Therefore the use of osmotolerant yeasts and ethanol superproducers is very desirable in the production of ethanol.

The fermentation of the must is a complex microbiological process that involves interactions between yeasts, bacteria and filamentous fungi (Aranda A, Matallana E, Olmo M. 2005. Yeasts. Saccharomyces I. First fermentation yeasts. In Wine Microbiology, Carrascosa AV Coordinators , R. Muñoz and R. González, pp 19-56. AMV Ediciones, Madrid). Traditionally, the fermentation of the must has been a spontaneous phenomenon, carried out by the microbiota that is found on the surface of the grape grain and in the cellar. However, this microbiota can be affected by a large number of factors, such as sanitary conditions, climatic conditions, degree of maturity of the crop, grape variety, use of phytosanitary products, etc. Therefore, the yeasts present in the fruits are not constant throughout the years, nor are their metabolic characteristics that are, to a large extent, responsible for the organoleptic characteristics of the wine obtained. This circumstance gives rise to an unwanted variation in production from year to year. To solve this problem and also in an attempt to obtain a product similar to that produced in traditionally producing regions, countries with a new wine tradition began fermentation with selected yeast inoculums and imported from countries with deep-rooted tradition. It has been proven that the use of selected yeasts has important advantages over traditional spontaneous fermentation. However, it is impossible to obtain a good strain for any type of winemaking, thus, yeasts from different producing regions have been obtained in order to obtain selected yeasts better acclimatized to each area in question. Today, the use of active dry yeast (LSA) selected to solve different problems is increasingly being imposed. In general, selected wine yeasts should be able to produce quality wines. And this quality must be maintained for the same type of must and preparation procedure, avoiding the variability and problems that unwanted wild microorganisms can cause. The use of selected yeasts in winemaking processes has basically five advantages over traditional spontaneous fermentation (Melero R. 1992. Controlled fermentation and selection of wine yeasts. Rev. Esp. Cienc. Tecnol. Aliment. 32, 371-379), which are: higher fermentation speed, maximum consumption of reducing sugars, greater reproducibility in the quality of the wines, reduction of the problems caused by wild wild yeasts, greater control of the sensory quality of the wine.

The criteria for selecting yeasts for use in oenology are: low production of volatile acidity, total consumption of fermentable sugars, high fermentative vigor and maximum reduction of latency phase, fermentation capacity at high and low temperatures, resistance to sulfur dioxide, low sulfur dioxide production, low hydrogen sulfide production, high sedimentation rate and flocculation capacity, low foam production, killer phenotype, low acetaldehyde production, adequate glycerol production, ethyl acetate production and higher alcohols, assimilation of nitrogenous substances , synthesis and excretion of amino acids and amino derivatives, production of β-glucosidase, low production of ethyl carbamate precursors. Currently, there are more than 200 well-characterized commercial yeast strains available in the market (Schuller D, Casal M. 2005. The use of genetically modified Saccharomyces cerevisiae strains in the wine industry, Applied Microbiology and Biotechnology 68 (3), 292 -304). Currently, the research is mainly directed in three ways: a) isolation of new local strains, better acclimated to each producing region, which allow to meet the needs of each winemaker, b) obtaining strains with specific interesting properties such as: flocculant strains, which Separate better from wines by facilitating clarification, strains with β-glucosidase activity, capable of releasing terpenes responsible for varietal primary aromas that improve the organoleptic characteristics of certain wines and c) introduction of specific genetic markers in the selected strains to determine and control their growth in non-sterile musts (Degré R. 1993. Selection and commercial cultivation of wine yeast and bacteria, in Wine Microbiology and Biotechnology, edited by GH Fleet, pp. 421-447, Harwood Academy Publishers).

So, although there are quite a few LSAs commercial, theoretically appropriate for vinifications, it is much more appropriate to use native (local) strains selected, since they ensure its implantation as a biological agent most important responsible for alcoholic fermentation, in addition to maintain the typical characteristics of the local wines (Suarez JA, Iñigo B. 2003. Oenological Microbiology. Fundamentals of Winemaking Mundi-Prensa editions, Madrid). This It is accentuated in those areas of warm climates where yeasts they are adapted to the high concentration of sugars, ethanol and high temperatures.

In the patent "Process of sequential directed fermentation, new strain of yeast involved in it, and its industrial application" (ES 2 222 786 A1) refers to a process of sequential directed fermentation, by which the must is sown at different times for the CECT 11773 strain of Pichia fermentans and for another of the Saccharomyces genus. The first results in the synthesis of a large amount of aromatic and flavoring substances with low ethanol production, which will determine the aroma of the final product; The second yeast is responsible for finishing the fermentation by increasing the amount of alcohol accumulated up to 12-13% v / v. The Enology application of selected wine yeasts simplifies, from the microbiological point of view, the wine fermentation process and improves its control.

Bioethanol is the ethyl alcohol produced from the fermentation of sugars found in plant products (cereals, sugar cane, beets, etc.) combined in the form of sucrose, starch, hemicellulose and cellulose. Mainly, the microorganism used to carry out this process is S. cerevisiae . Currently, there are numerous studies focused on the production of ethanol as fuel from renewable sources as an economically viable alternative to gasoline. Most studies focus on improving the efficiency of the process by increasing the speed of ethanol production, the latter is limited by the ethanol producing microorganism, due to its toxic effect (Allain EJ. 2007. Cell- free ethanol production: The future of fuel ethanol. J Chem Technol Biotechnol 82 : 117-124). In this sense, there are numerous research projects aimed at isolating and selecting different wild strains of yeasts obtained from various substrates such as soil, ripe fruits, molasses, fermented foods, etc. (Kiran Sree N, Sridhar M, Suresh K, Banat IM, Venkateswar Rao L. 2000. High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae . 2000. J Ind Microbiol Biotechnol 24 : 222-226; Brooks AA. 2008 Ethanol production potential of local yeast strains isolated from ripe banana peels Afr J Biotechnol 7 : 3749-3752; Gomes FCO, Silva CLC, Marini MM, Oliveira ES, Rosa CA. 2007. Use of selected indigenous Saccharomyces cerevisiae strains for the production of the traditional cachaca in Brazil. J Appl Microbiol 103 : 2438-2447), there being in recent years a large number of patents in aspects related to ethanol producing organisms. New genetically improved strains have also been obtained (Knauf M, Kraus K. 2006. Specific yeast developed for modern ethanol production. 2006. Sugar Industry 131 : 753-758; Seki T, Myoga S, Limtong S, Uedono S, Kumnuanta J, Taguchi H. 1983. Genetic construction of yeast strains for high ethanol production, Biotechnol Lett 5 : 351-356). An ideal microorganism to produce ethanol must have a good and fast fermentative potential, flocculability, appreciable osmotolerance, high ethanol tolerance and good thermotolerance (Knauf and Kraus, 2006, Chandel AK, Narasu ML, Chandrasekhar G, Manikyam A., Rao LV. 2009. Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS_ {3}. Biores Technol 100 : 2404-2410).

Explanation of the invention.

As indicated above, the fermentation of grape must with high sugar content for the production of sweet wines presents a series of technical problems. A possible solution could be the realization of fermentations directed with isolated osmotolerant yeasts and selected from these types of musts. In the present invention, a natural yeast from a Pedro Ximénez grape must has been isolated and selected with an initial concentration of reducing sugars of 450 g / L. It is a strain of Saccharomyces cerevisiae , CECT 13015, and is characterized in that it is osmotolerant and a good producer of ethanol. Experiments of microvinification directed with this yeast demonstrates a faster fermentation kinetics and an improvement of the organoleptic characteristics of these wines, with respect to those obtained by spontaneous fermentation. The use of this wine yeast in the wine industry could be useful to improve the aroma of wines, especially sweet wines.

On the other hand, in a second aspect of the present invention and due to the growing demand for ethanol by the energy crisis of fossil fuels, the search for osmotolerant and high production yeast strains of ethanol The strain CECT 13015 shows greater growth and Fermentation kinetics in all fermentations tested. Microfermentation experiments indicate that this yeast is of Useful use in the production of bioethanol from different sugary substrates such as molasses or juices obtained from beet or others.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

Brief description of the drawings

Fig. 1 shows the evolution of the volumetric mass (g / L) during yeast fermentation osmo-tolerant ethanol.

Fig. 2 shows the characteristics of the wines of Pedro Ximénez raisins where (A) shows the variables common oenological, (characteristics of the wines), being 1: Ethanol (% v / v), 2: A. volatile (g / l), 3: A. total (g / l). 4: pH v 5: d (q / ml) (B) Major higher alcohols, (C) Compounds carbonyl and (D) major polyalcohols, where 6: wine traditional, 7: control and 8: yeast CECT 13015.

Detailed presentation of embodiments and examples

The present invention relates to the isolation and selection of a strain of Saccharomyces cerevisiae , CECT 13015, for application in fermentations directed for the production of sweet wines as well as for obtaining bioethanol from sugary substrates.

Isolation and selection of osmotolerant strains

Different osmotolerant strains of Saccharomyces cerevisiae were isolated from a fermented must of sun-dried grapes (450 g / L of initial fermentable sugars) by the seeding procedure by depletion in Petri dishes with YPD agar (1% yeast extract , 2% peptone and 5% glucose, pH 5.5). The boxes were incubated at 28 ° C for 48 h. Subsequently, each colony was passed to a tube with inclined YPD agar to obtain a pure culture. The pure cultures were stored in a refrigerator at 4 ° C or at -80 ° C until use. A strain has been selected for its osmo-ethanol-tolerance characteristics, good organoleptic characteristics in musts with high sugar content as well as for ethanol production. The identification and deposit of the yeast strain was carried out by the Spanish Type Culture Collection (CECT), identifying with the number CECT 13015.

Wort, preferential corrections, conditions of fermentation and inoculation to obtain sweet wines

The initial grape must Pedro Ximénez passes had a volumetric mass value of 1.1543 ± 0.0015 g / mL, equivalent to a content in reducing sugars of 373 ± 4 g / L and approximately 22% (v / v) of potential ethanol. The content in Titratable acidity was 3.31 ± 0.07 g / L, volatile acidity 0.07 ± 0.02 and the pH had a value of 4.30 ± 0.02.

The must was corrected to a pH value of 3.8 by the addition of tartaric acid and 50 mg / L of S02 was added as potassium metabisulfite and was divided into 6 750 mL fractions which were placed in individual 1L specimens. Three specimens were they were destined for an unfermented control, and 3 for strain CECT 13015. The specimens were covered with hydrophobic cotton and immersed in a thermostated bath with water at 24ºC.

Prior to inoculation, the S. cerevisiae strain CECT 13015 was grown separately in YPD medium for 24 hours at 28 ° C and subsequently collected by centrifugation and washed with sterile and cold distilled water. The inoculum of each yeast for each test tube with must was 6 ± 1x10 6 cells / mL.

Ethanol production

The yeast cell was cultured in YPD medium (1% yeast extract, 2% peptone and 5% glucose, pH 5.5) autoclaved. The cells were incubated at 24 ° C under continuous agitation at 200 rpm in a Shaker incubator (New Brunswick Scientific, UK) for 24 h and collected by centrifugation at 3500 x g .

The microfermentations were carried out at 24 ° C in 50 mL Falcon tubes with 20 ml of the 1% extract solution of yeast, 2% peptone and increasing glucose concentrations of 25 to 45% (w / v). The fermentation media were steam sterilized fluent for 30 minutes. The fermentation media were inoculated with 4x10 6 cells / ml. The plugs of the Falcon tubes are they pierced with a hypodermic needle and stirred at 150 rpm.

The total cell number was determined by counting in an electronic particle counter (Beckman Coulter Z2 Particle Count and Size Analyzer). The development of the fermentation process was monitored by the amount of CO2 released as a measure of weight loss (Sablayrolles et al ., 1987; Bely et al ., 1990).

Ethanol was quantified by the Enzymatic Kit of Boehringer-Mannheim, Germany.

Example 1 Obtaining sweet wine

Fig. 1 shows the fermentation kinetics. The must inoculated with strain CECT 13015 showed a mass value 1078 g / L volume at 140 hours.

The final ethanol content was 12% (v / v) of ethanol At this time the fermentation was stopped by addition of wine alcohol to an ethanol content of 15% (v / v) and a volumetric mass of 1062 g / L for CECT 13015. On the other hand, the control corresponds to the addition of wine alcohol to the initial must up to a value of 10% (v / v) and a value of 1153 g / L was obtained, which It was not altered throughout the trial.

The average values are shown in Fig. 2 of the compounds and fractions quantified in each of the experiments performed and in a sample of PX wine made from the traditional way. In Fig. 2A it is noted that musts partially fermented with CECT 13015 presented values of the volatile acidity superior to those of the two remaining musts unfermented

Fig. 2B shows the concentration of alcohols Majority superiors and methanol, highlighted the increased production of the same in relation to the other musts. Among the compounds quantified carbonyls (Fig. 2C), highlighted the content in acetoin of control wort (unfermented) and acetate content of ethyl wine made in the traditional way. Refering to production of 2,3-butanediol and glycerin (Fig. 2D) both compounds had a higher content in musts fermented with CECT 13015.

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Example 2 Obtaining Bioethanol

one

The medium used to study the production of Ethanol contained 25 to 45% (w / v) glucose. The results exposed in the previous table show that the strain CECT 13015 could be used to obtain ethanol due to its high efficiency

Claims (6)

1. Wine yeast belonging to the Saccharomyces cerevisiae species deposited in the Spanish type crop collection, with identification number CECT 13015. 2. Wine yeast according to claim 1 characterized in that it has an osmotic tolerance between 25-45% (w / v) glucose and an ethanol tolerance between 12-15% (v / v). 3. Wine yeast according to the preceding claims characterized in that it produces a maximum of 20% (v / v) ethanol at 28 ° C. 4. Use of wine yeast from claims 1 to 3 for the production of beverages alcoholic 5. Use of wine yeast from claims 1 to 3 for the production of sweet wines natural 6. Use of wine yeast from claims 1 to 3 in the production of ethanol from sugary substrates by inoculation to the medium of 4x10 6 cells / mL of said yeasts.